The major histocompatibility complex (MHC) plays a key role in the defense mechanism of a body induced by T cell immunoresponses by presenting a cancer- or virus-derived antigen peptide. MHC is classified as class I or class II, and class I is expressed in all somatic cells except for germ cells and erythrocytes. MHC class I is composed of an α chain and a β chain, and the α chain is composed of α1 and α2 regions associated with the formation of an antigen peptide-holding groove and an α3 region associated with the binding to a co-receptor CD8 molecule expressed on the cytotoxic T cell (CTL) surface. In contrast, the β chain of MHC class I is encoded by β2 microglobulin gene (Non-Patent Document 1).
The cytotoxic T cell (CTL) is known to be activated by recognizing a complex of an MHC class I molecule and an antigen peptide existing on the antigen presenting cell (APC) surface with the aid of a T cell receptor and it is known to specifically damage cancer cells or virus-infected cells expressing the same complexes. With the use of such cell-mediated immune mechanism, the development of vaccines used for cancer immunotherapy or treatment of infections has been attempted.
MHC class I (H-2 class I) of mice that are commonly used as experimental animals is different from MHC class I (HLA class I) of humans. Thus, mice cannot be used as in vivo evaluation models for the development of human vaccines. Thus, human HLA genes containing a promoter region or recombinant genes comprising the HLA genes inserted into sites downstream of a mouse H2 promoter region have been injected into fertilized mouse eggs, so as to prepare various transgenic mice expressing human HLA class I molecules (Non-Patent Documents 2 and 3). However, such transgenic mice were not capable of sufficiently recognizing the α3 region of human HLA class I with the use of mouse CD8. Thus, HLA-restricted CTL induction was limited. In order to overcome such problems, a chimeric gene comprising HLA class I α1/α2 fused to mouse H-2 class I α3 was inserted into a site downstream of SV 40 promoter to construct transgenic mice (Non-Patent Document 4), and in vivo evaluation of HLA-restricted CTL induction became feasible for the first time.
In such transgenic mice, however, endogenous mouse H-2 class I is expressed. When mouse CTL recognizes an antigen, it preferentially uses the H-2 class I molecule. Thus, HLA class I-restricted CTL induction was incomplete. In order to overcome this problem, double knockout mice for mouse H-2 class I α molecules (H-2Kb and H-2Db) were subjected to crossing with transgenic mice comprising HLA class I chimeric genes, so as to construct combined mutant mice and improve HLA class I-restricted CTL induction (Non-Patent Document 5).
The β2 microglobulin gene constituting the MHC class I β chain is essential for the MHC class I molecule to function. Thus, functions of mouse MHC class I are lost in β2 microglobulin gene-deficient mice (Non-Patent Documents 6 and 7). In addition, a single-chain fusion gene resulting from the binding between the C terminus of β2 microglobulin and the N terminus of MHC class I with the aid of a peptide linker consisting of 15 amino acids is known to function normally in cells (Non-Patent Document 8).
An artificial MHC class I gene comprising β2 microglobulin, HLA class I α1 and α2 domains, and the mouse H-2 class I α3 domain fused to each other was inserted into a site downstream of HLA class I gene promoter, so as to construct transgenic mice. The resulting transgenic mice were subjected to crossing with the β2 microglobulin gene knockout mice, so as to construct combined mutant mice (Non-Patent Document 9). In such mice, H-2 class I expression was substantially lost and HLA class I-restricted CTL induction reaction was observed. Accordingly, such mice are considered to be the most preferable MHC class I humanized mouse at present.
In recent years, the MHC class I humanized mouse was subjected to crossing with more severely immunodeficient animals (i.e., NOD/SCID/gamma-c-null (NOG) mice), and human blood stem cells were implanted into the resulting multiple mutant mice. Thus, human-adaptive immune systems having complete HLA-restricted T cells can be reconstructed (Non-Patent Document 10). By subjecting the MHC class I humanized mouse to crossing with other mutant lines, the resulting mice may become useful for the production of superior animal models of human immune systems.
However, transgene expression in artificial MHC class I gene transgenic mice that have been constructed in the past is considered to be influenced by various factors such as copy number, site of insertion in the genome, or epigenetic modification. This required the selection of mouse lines showing optimal expression levels from among constructed transgenic mouse lines. For the same reasons, it has been difficult to compare the capacity of CTL induction among transgenic mouse lines expressing different MHC class I genes.
In order to enhance human MHC class I-restricted CTL induction, it has been necessary to subject the selected mouse lines to crossing with the β2 microglobulin gene deficient mice. Accordingly, additional time and labor has been required, after selection of transgenic mouse lines showing the optimal expression levels.
While the MHC class I humanized mouse has been very useful, the transgenic mouse lines thereof that have been heretofore constructed have been limited to A1, A2, A24, A11, B44, and B7 for the reasons described above. The production of combined mutant mice via crossing of the MHC class I humanized mouse and the β2 microglobulin gene knockout mice has been further limited.